Reversible RNA Acylation Using Bio-Orthogonal Chemistry Enables Temporal Control of CRISPR-Cas9 Nuclease Activity

The CRISPR–Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a trans-cyclooctene (TCO)–acylimidazole reagent that acylates 2′–OH groups of RNA. Poly acylation (“cloaking”) of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-step process (“uncloaking”). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism’s development.


Table of Contents Figure and Scheme titles Page
Materials and Methods S2-S4 Evaluation of kinetics of the reaction between (2E)-2-cyclooctene-1-carboxylate and Tz

S5
Table S1 Predicted MALDI peaks (negative mode) of the 18-nt model RNA.

MATERIALS AND METHODS
All oligonucleotide solid phase syntheses were done on a 1.0 μmol scale using the Oligo-800 synthesizer (Azco Biotech, Oceanside, CA, USA).Solid phase syntheses were performed on control-pore glass (CPG-1000) purchased from Glen Research (Sterling, VA, USA).Other oligonucleotide solid phase synthesis reagents were obtained from ChemGenes Corporation (Wilmington, MA, USA).Phosphoramidites (TBDMS as the 2′-OH protecting group): rA was N-Bz protected, rC was N-Ac protected and rG was N-iBu protected.A, C, G, U phosphoramidites were dissolved in anhydrous acetonitrile (0.07 M) directly before use.m 1 A, m 6 A, s 2 U and s 4 U phosphoramidites were dissolved in anhydrous acetonitrile (0.15 M) directly before use.Coupling step was done using 5-ethylthio-1H-tetrazole solution (0.25 M) in acetonitrile for 12 min.5′detritylation step was done using 3% trichloroacetic acid in CH 2 Cl 2 .Oxidation step was done using I 2 (0.02 M) in THF/pyridine/H 2 O solution.
For gel electrophoresis, 10X Tris/Borate/EDTA (TBE) buffer was purchased from Fisher Scientific Company L.L.C. (Waltham, MA, USA) and used with proper dilution.30% Arcylamide/Bis-arcylamide solution (29:1) was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).GeneRuler 1 kb Plus DNA Ladder (cat.#FERSM1331) was purchased from Fisher Scientific.Chromatographic purifications of synthetic materials were conducted using SiliaSphere TM spherical silica gel with an average particle and pore size of 5 μm and 60 Å, respectively (Silicycle Inc, QC, Canada).Thin layer chromatography (TLC) was performed on SiliaPlate TM silica gel TLC plates with 250 μm thickness (Silicycle Inc, QC, Canada).Flash chromatography was performed using Biotage Isolara One instrument (Biotage Sweden AB, Uppsala, Sweden).Preparative TLC was performed using SiliaPlate TM silica gel TLC plates with 1000 μm thickness.C NMR spectra were proton decoupled.High resolution ESI-MS spectra of small molecules was acquired using Agilent Technologies 6530 Q-TOF instrument.MALDI-TOF experiments were performed at the Stanford University Mass Spectrometry facility, using Bruker Daltonik Microflex MALDI-TOF spectrometer equipped with an N2 laser.RNA samples were plated on an MSP Anchorchip 96 target plate and mass spectra were recorded in linear negative mode.Matrix consisted of 0.3 M trihydroxyacetophenone in EtOH and 0.1 M aqueous ammonium citrate, which were mixed in a 2:1 ratio by volume.Kinetic experiments were carried out using CARY RNA Sequences: Capital letters indicate unmodified nucleotides, while small letters correspond to nucleotides containing 2′-OMe groups.

Kinetics Experiments
The kinetics of the reaction between (2E)-2-cyclooctene-1-carboxylate and Tz was monitored by UV-Vis spectroscopy at 520 nm.This experiment was conducted under pseudo 1 st order conditions in a 1:1 solution of DMSO:PBS (pH 7.4) at 25 °C with concentrations of (2E)-2cyclooctene-1-carboxylate (5 mM) and Tz (0.5 mM).Absorbance at 520 nm was measured every 10 seconds.The kinetic experiments were performed in triplicate.Data were analyzed in GraphPad Prism 10 Software.The observed pseudo 1 st order rate constant was used to calculate the second order rate constant.

Un-Cloaking Procedure:
Uncloaking was carried out by combining 1 μL of Tz (1 mM in PBS) and 1 μL of cloaked sgRNA 1 or sgRNA 2 (5 μM in H2O ).The solution was placed in a thermoshaker at 37 ˚C for 2 h.After that, the uncloaked sgRNA 1 or sgRNA 2 were used directly for CRISPR experiments.

CRISPR-Cas9 experiments in HEK293 cells:
Cloaked sgRNA CRISPR-Cas9 experiments, were carried out following the procedure reported by Yin, H. et al. [Nat. Chem. Biol. 2018, 14, 311-316].The GFP-expressing HEK293 cells were purchased from GenTarget (cat# SC001) and cultured in DMEM, containing 10% FBS and 1X Penicillin/Streptomycin, at 37 °C, 5% CO2, and 95% humidity.The cells were seeded at a concentration of 1 × 10 5 cells per well in 6-well plate 24 h prior to the experiment.The cells were transfected with Cas9 mRNA (500 ng, Thermo Fisher Scientific), GFP-targeting sgRNA 2 (30 nM) or cloaked sgRNA 2 (30 nM) using lipofectamine (1.5 µL) (Invitrogen™ LMRNA003) for 72 h in Opti-MEM reduced serum media.After 72 h, Opti-MEM was replaced with fresh DMEM and the cells were grown for additional 48 h.The cells were treated with trypsin for 5 min, collected by centrifugation at 1000 RPM and suspended in PBS (1 mL).GFP expression was analyzed by flow cytometry.Data from 10 6 cells were acquired using a FACS Aria III cell sorter equipped with a 488 nm/blue coherent sapphire solid-state laser, 20 mW (BD Biosciences, San Jose, CA, USA).Data analyses were carried out using FlowJo software (Ashland, OR, USA), according to manufacturer's instructions.Parameters, such as MFI and the percentages of specific populations were quantified by histogram analysis.

Figure S1 .
Figure S1.Evaluation of kinetics of the reaction between (2E)-2-cyclooctene-1-carboxylate and Tz performed in 1:1 solution of PBS (pH 7.4) and DMSO.Plot of absorbance at 520 nm, as function of time.The experiments were performed in duplicate.The shown data is representative of the duplicate experiments.

Figure S2 .
Figure S2.MALDI-TOF spectrum of the model RNA cloaked with TCO-Im for 1 h.

Figure S3 .
Figure S3.MALDI-TOF spectrum of the model RNA cloaked with TCO-Im for 2 h.

Figure S4 .
Figure S4.MALDI-TOF spectrum of the model RNA cloaked with TCO-Im for 4 h.

Figure S5 .
Figure S5.Uncloaking of model RNA with Tz for 1 h.

Figure S6 .
Figure S6.Uncloaking of model RNA with Tz for 2 h.

Figure S7 .
Figure S7.Viability of GFP-expressing HEK293 cells treated with variable concentrations of Tz.All experiments were performed in six replicates.Error bars represent ± s.d.

Figure S8 .
Figure S8.Flow cytometry analysis of CRISPR experiments targeting the GFP gene in GFPexpressing HEK293 cells.The experiments were performed in duplicate.The shown data is representative of the duplicate experiments.(A) Histograms of total GFP-expressing cells: untreated cells are shown in green, cells transfected with the sgRNA2 are shown in grey.(B) Histograms of total GFP-expressing cells: untreated cells are shown in green, cells transfected with cloaked sgRNA2 are shown in grey.

Figure S9 .
Figure S9.Flow cytometry analysis of GFP-expressing HEK293 cells treated with different concentrations of Tz.The experiments were performed in duplicate.The shown data is representative of the duplicate experiments.

Table S1 .
Predicted MALDI-TOF peaks (negative mode) of the 18-nt model RNA.Predicted peak heights are shown in the parentheses.
Compound 1 was synthesized by following the reported procedure[Org.Lett.2020,22, 6041- 6044].Compound 1 was isolated as a mixture of axial and equatorial (eq) isomers in a 1:2.5 ratio.The obtained NMR spectra matched the reported ones.Dissolved 1 (100 mg, 0.65 mmol) in 162 µL of DMSO-d6 to prepare a 4 M solution.Prepared a suspension of CDI (126 mg, 0.78 mmol) in 162 µL of DMSO-d6.Dropwise added the suspension of CDI to the solution of 1 in DMSO-d6.Agitated the reaction mixture for 5 min until the evolution of CO2 was complete.The resulting 2 M DMSO solution of the crude product was used for RNA cloaking experiments without further purification.NMR spectra is a mixture of of axial and eq TCO isomers in a 1:2.5 ratio, plus one equivalent of imidazole: